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Supplemental Online Materials

Materials and Methods
Clinical response and autoimmunity assessment
Response was assessed by comparison of radiographic measurements and physical
examination findings before and after treatment. A complete response was defined as the
complete disappearance of all evaluable disease. A partial response was defined as a decrease
equal to or greater than 50 percent in the sum of the products of perpendicular diameters of all
measurable lesions for at least one month. A mixed response was defined as a decrease in the
area of some lesions with concurrent growth of other lesions or the appearance of new lesions.
Autoimmunity was assessed by physical examination for skin depigmentation (vitiligo) and
ophthalmic examination (uveitis).

Derivation of cells used for treatment

T cell cultures for infusion were derived from tumor infiltrating lymphocytes (TIL) by
minor modifications of established techniques 1;2. Each patient had one or more deposits of
metastatic melanoma excised, and multiple independent TIL cultures were started from each
nodule. TIL cultures were initiated by the explant of a small (2mm3) tumor fragments or by
plating 1 x 106 viable cells of a single cell suspension of enzymatically digested tumor tissue into
2 ml of complete medium (RPMI1640 based medium supplemented with 10% human serum)
containing 6000 IU/ml of IL-2. The cultures were maintained at cell concentrations between 5 x
105 and 2 x 106 cells per ml until several million TIL cells were available, usually 2-4 weeks.
Multiple independent cultures were screened by cytokine secretion assay for recognition of
autologous tumor cells (if available) and HLA-A2+ tumor cell lines. Two to six independent TIL
cultures exhibiting the highest cytokine secretion were further expanded in complete medium
with 6000 IU per ml IL-2 until the cell number was over 5 x 107 cells (this cell number was
typically reached 3-6 weeks after tumor excision). TIL cultures that maintained specific tumor
cell recognition were expanded for treatment using one cycle of a rapid expansion protocol 3
with irradiated allogeneic feeder cells, OKT3 (anti-CD3) antibody, and 6000 IU per ml IL-2.
This rapid expansion protocol typically resulted in 1000-fold expansions of cells by the time of
administration 14-15 days after initiation of the expansions.

Analysis of TCR expression by FACS, RT-PCR and nucleotide sequencing.

T cell receptor (TCR) beta-chain variable region (Vβ) frequency was determined using
two color FACS with a FITC-conjugated CD8-specific antibody and a panel of PE-conjugated
Vβ specific antibodies (Beckman/Coulter/Immunotech, except FITC-labeled Vβ5a, Vβ5c and
Vβ6.7 from Pierce/Endogen). Cryopreserved samples were thawed, Fc receptors were blocked
by incubation with mouse Ig, and cells were stained with the indicated antibodies. Cells were
washed twice and analyzed using a Becton Dickenson FACStar with Cellquest software. A
lymphocyte gate was established based on forward and side scatter profiles, and dead cells were
excluded based on propidium iodide staining. The percentage of Vβ+ cells in the CD8+ fraction
was calculated as 100 times the ratio of dual positive (PE+/FITC+) cells to all CD8+ (FITC+)
cells. A skewed Vβ family was defined as one that comprised more than two percent of the total
CD8+ cells and was more than 4 fold higher than the median value of all Vβ families analyzed in
the sample. HLA-A2/MART-1 tetramer staining was carried out according to the
manufacturer’s recommendations (Beckman/Coulter/Immunomics). The MART-1 specific Vβ7+

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cells from patient 10 were not analyzed with the HLA-A2/MART-1 tetramer because that
tetramer is made with the MART-1:26-35(27L) heteroclitic peptide and patient 10’s Vβ7+ cells
recognized the native MART-1:27-35 peptide, but do not recognize the 27L-modified peptide.
Because the panel of Vβ family-specific antibodies failed to recognize about one third of
the expressed TCR Vβ families, additional analysis of TCR expression in TIL and peripheral
blood lymphocytes (PBL) was undertaken using RT-PCR with PCR primers that were designed
to amplify all Vβ gene families 4. Seven days after cell transfer strong RT-PCR products were
seen in PBL from patient 9 for the reactions with Vβ12 and Vβ14 primers, and in PBL from
patient 10 eight days after TIL transfer only with the Vβ7 primers 5.
To assess the diversity of the TCR within the over represented Vβ families, the
nucleotide sequence of the β-chain V-D-J regions was determined. The Vβ12 specific RT-PCR
products from patient 9 were cloned by ligation into a T/A cloning vector. Six DNA clones from
PBL and six clones from TIL were used as templates for sequence analysis of the TCR V-D-J
junction by PCR cycle sequencing. All these clones were found to have the identical nucleotide
sequence, which was also identical to the V-D-J sequence from a MART-1 specific T cell clone
derived from the TIL. In contrast, none of six DNA clones from the VB12 specific RT-PCR
products of pretreatment PBL shared any V-D-J junctional sequence homology with the MART-
1 specific TCR sequence. Further sequence analysis was undertaken using the RT-PCR products
excised from agarose gels as the sequencing template. Using this approach, Vβ specific RT-PCR
products from pretreatment samples generated unreadable sequence information due to the
heterogeneous TCR composition of the template pool. In contrast, the VB12 specific RT-PCR
products of the infused TIL, post-treatment PBL, or tumor specimens biopsied 20 days after cell
infusion from patient 9 showed a single V-D-J junctional sequence identical to the sequence
derived from the TIL clones. Similar nucleotide sequence analysis with Vβ7 specific RT-PCR
products from the infused TIL or post infusion PBL from patient 10 generated a single clear
sequence through the V-D-J junctional region, and the sequence was identical to that from a
MART-1 reactive Vβ7+ T cell clone derived from the TIL.

Cytokine release assay and cytotoxicity assay

To determine specific cytokine release, effector cells (1 x 105) were washed twice and cultured
overnight with stimulator cells (1 x 105) in duplicate wells of a 96 well plate. Stimulator cells
included tumor cell lines and the TAP deficient T2 cell line pulsed with HLA-A2 peptide
epitopes gp100:209-217 or MART-1:27-35. Coculture supernatants were assayed for IFN-γ
release by ELISA assay (Pierce/Endogen). Specific cytokine release was defined as values
greater than 100 pg/ml and at least twice all background values.
Lymphocyte mediated cytotoxicity was determined by a standard Cr-release assay.
Briefly, T cells were incubated with 51Cr-labelled target cells at the indicated effector:target ratio
for four hours and 51Cr levels in the supernatant were measured. Specific lysis was calculated by
the formula: 100 x (experimental release - spontaneous release)/maximum release – spontaneous
release).

Immunohistochemistry of resected tumor specimens.

For analysis of TCR expression in situ, tumor specimens were resected from, embedded in OCT
compound, and cryopreserved. Cryosections were stained with a panel of Vβ antibodies and
counterstained with Eosin.

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For analysis of T cell phenotype and MHC expression, resected tumor specimens were fixed
embedded in paraffin, and sectioned. Slides were stained with the indicated antibodies on an
automated immuno-stainer according to standard techniques and the manufacturer’s
recommendations.

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Figure S1. Antitumor and autoimmune manifestations following the transfer of

lymphocytes with anti-MART-1 reactivity. Upper: CT scan of the lower extremity of patient 9
showing multiple melanoma metastases (arrow) in the skin and subcutaneous tissue (left) that
underwent regression (right) after cell transfer. Middle: As cancer regressed in the lower
extremity of patient 9, patchy areas of vitiligo appeared as shown on the forearm. Lower: Two
weeks after cell transfer, as melanoma nodules were regressing, patient 10 experienced decreased
vision caused by an autoimmune uveitis resulting in a fibrinous pupillary membrane with
extensive iris adhesions to the lens (arrow) that cleared following the administration of steroid
eye drops.

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Table S1: Activity and specificity of infused lymphocyte cultures (IFN-γ pg/ml). All samples for infusion demonstrated
significant cytokine release when stimulated with autologous or HLA-A2+ tumor cell lines. Six infusion samples recognized MART-
1, five recognized gp100, and three recognized unidentified antigens expressed by the autologous tumor cell line. There was no
correlation between the antigen specificity of the infused cells and objective response, the onset of autoimmunity, or treatment
toxicity.

Stimulation by tumor lines (HLA-A2)2 Stimulation by 293-A2 gene transfectants3

1
Patient None 938 (-) 526 (+) Autol. (+) GFP MART-1 TRP1 TRP2 TYR NY-ESO-1 gp100 MAGE1 MAGE3

1 204 143 155 5140 155 151 123 137 126 128 110 133 151
2 9 7 9850 7020 0 6850 53 0 0 65 950 0 0
3 1 3 20420 615 0 0 0 0 2 0 7100 0 0
4 0 4 9090 nd 0 0 75 0 0 0 4390 0 0
5 0 2 19 3535 0 0 0 0 2 0 0 0 0
6 0 9 798 2620 68 121 94 87 121 104 118 96 104
7 0 279 5170 5230 0 3790 0 0 0 0 28 23 83
8 0 849 4990 nd 0 0 0 2 0 0 458 0 0
9 11 43 2528 nd 12 >1361 17 13 17 30 17 14 19
10 0 0 15150 nd 0 13360 13 0 0 0 20 0 33
11 0 0 9025 nd 0 12110 10 0 7 65 287 0 70
12 11 72 1200 1006 126 1970 120 80 96 60 145 88 209
13 0 234 3545 703 75 1730 0 0 0 0 19 0 0
1
Τhe results in this table are combined from three different experiments. Values indicate pg/ml of IFN-γ. Significant T cell reactivity was defined by values that
are at least two times all controls and greater than 100 pg/ml (bold and underlined).
2
Melanoma cell lines were derived from tumor specimens obtained at the Surgery Branch, NCI. If a patient had no autologous melanoma cell line available at
the time of the assay, the value is listed as “nd” (not done).
3
The human embryonic kidney cell line 293 was first stably transfected with an expression construct for HLA-A2, then ransiently transfected with a panel of
plasmids encoding shared melanoma antigens. Efficient transfection was confirmed in each experiment by visual assessment of green florescent protein (GFP)
expression in GFP transfected cells and in some assays by the stimulation of T cell clones by the appropriate transfectants including MART-1, tyrosinase related
protein (TRP)2, tyrosinase (TYR), NY-ESO-1, and gp100.

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Table S2: T cell receptor Vβ gene usage in CD8+ cells from TIL and post treatment PBL.
To investigate the function and fate of the transferred T cell populations, T cell receptor (TCR)
expression was examined using a panel of beta chain variable region (Vβ) specific antibodies
in all patients for whom peripheral blood samples were available at one week and approximately
one month post cell transfer. Vβ expression was highly skewed (underlined and bold) in five of
the six administered TIL, and some of these same Vβ families were also over represented in the
peripheral blood of the patients at one week after cell transfer, suggesting that those T cells in the
PBL were derived from the infused TIL (boxed). Patients 9 and 10 demonstrated a persistent
high level engraftment of individual T cell clones.
Vβ family (percent of CD8+ cells)
Patient Day1 CD82 1 2 3 5a 5c 6.7 7 8 9 11 12 13 14 16 17 18 20 21 22 23
6 Pre 1222 6 12 1 3 1 2 3 4 2 3 1 3 5 1 4 0 3 3 2 2
TIL - 12 7 0 6 0 2 2 0 0 0 0 2 2 0 5 0 19 1 1 1
8 115 4 11 1 4 0 2 1 4 0 0 1 3 2 1 7 0 10 2 1 0
43 527 3 9 3 1 1 1 3 8 1 1 2 1 13 2 6 0 1 2 2 2
9 Pre 390 2 4 2 3 1 1 11 12 1 0 1 2 4 0 nd3 0 4 2 3 1
TIL - 0 1 0 1 1 1 0 0 0 0 14 0 2 0 nd 0 0 0 0 1
7 20150 0 0 0 0 0 0 0 0 0 0 63 0 8 0 nd 0 0 0 0 0
37 nd 0 0 0 1 0 0 0 0 0 0 69 0 6 0 nd 0 0 0 0 0
97 1090 1 0 1 0 1 1 1 0 0 0 82 0 6 0 0 0 0 0 4 0
123 593 1 0 4 1 1 1 1 1 1 0 72 0 8 0 0 0 0 0 4 0
10 Pre 1092 2 8 3 2 4 3 2 2 2 1 1 3 3 2 3 nd 0 1 2 1
TIL - 0 3 1 3 9 5 89 1 11 1 0 7 1 0 2 nd 1 0 0 1
7 11664 0 0 0 0 1 0 97 0 0 0 1 1 0 0 0 nd 0 0 0 0
23 3035 1 1 0 1 3 0 86 0 0 0 7 2 0 0 1 nd 2 0 1 0
30 2873 1 1 0 1 2 1 87 0 0 0 2 0 0 2 0 0 0 0 0 0
159 697 1 1 0 1 3 1 75 1 1 0 2 1 2 3 1 0 1 1 1 1
11 Pre 853 2 5 3 4 3 1 3 4 3 1 3 6 7 2 11 1 1 4 5 2
TIL - 0 2 89 4 4 4 3 1 0 0 1 0 6 0 0 0 0 0 0 3
6 298 0 4 57 1 1 0 0 0 2 2 3 2 14 1 1 2 3 3 3 3
39 81 2 6 3 6 1 1 2 1 0 0 5 3 3 2 32 2 2 7 4 0
12 Pre 787 5 6 2 3 2 1 5 3 2 2 2 3 3 2 3 0 1 2 3 2
TIL - 6 4 4 6 3 5 6 1 4 2 2 6 0 1 5 2 2 4 5 4
6 572 7 3 2 2 2 1 3 2 3 1 1 5 3 2 3 0 1 3 4 2
30 327 9 3 6 1 4 4 2 6 1 1 16 7 4 3 4 1 1 3 3 1
13 Pre 634 3 6 8 6 5 19 16 3 4 1 2 1 3 3 3 1 5 2 1 4
TIL - 1 6 1 3 3 4 5 3 1 0 1 2 2 0 5 0 0 0 50 20
8 400 1 2 1 1 1 1 1 7 0 0 1 0 2 0 3 0 0 1 15 43
15 nd 8 7 5 10 12 11 13 2 2 0 3 2 4 2 3 0 1 1 4 5
1
Pre treatment peripheral blood lymphocytes (Pre) were harvested prior to the start of the conditioning
chemotherapy. TIL were sampled prior to infusion. Post-infusion PBL were sampled on the indicated days after
cell transfer.
2
Absolute CD8 counts were calculated as the product of the absolute lymphocyte count on the indicated day and the
percent of CD8+ cells determined by FACS analysis.
3
not done.

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Reference List

S1. S. A. Rosenberg et al., J.Natl.Cancer Inst. 86, 1159-1166 (1994).

S2. M. E. Dudley et al., J.Immunother. 25, 243-251 (2002).

S3. S. R. Riddell and P. D. Greenberg, J.Immunol.Methods 128, 189-201 (1990).

S4. M. D. McKee, T. M. Clay, R. A. Diamond, S. A. Rosenberg, M. I. Nishimura,

J.Immunother. 23, 419-429 (2000).

S5. Unpublished observations.

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